Project Summary/Abstract The poly(ADP-ribose) polymerase (PARP) family comprises a diverse set of proteins with a conserved enzymatic activity that catalyzes the covalent attachment of ADP-ribose (ADPR) units from NAD+ molecules on target proteins. Initial studies of PARPs were focused primarily on the biochemistry and molecular biology of PARP-1 in DNA repair, but more recent studies have linked the major nuclear PARPs (PARPs 1, 2, and 3) to the control gene expression programs in a variety of signaling-regulated biological systems, including adipogenesis and inflammation. These studies have highlighted the role of PARP proteins in normal physiology, beyond their well-characterized role in disease states. Although progress has been made, PARP- related research must expand in several critical areas, with a greater emphasis on the following: (1) other PARPs besides PARP-1, (2) a broader view of PARP molecular functions, beyond DNA repair, (3) a broader view of PARP biology, beyond cancer and aging, (4) identification and evaluation of specific sites of ADP- ribosylation to better understand how the modification affects the activity of target proteins, (5) the importance of NAD+ metabolism and substrate availability, and (6) new methodologies to facilitate the study of PARPs. The long-term objective of these studies is to achieve a better understanding of the molecular and biochemical mechanisms underlying the control of signal-regulated transcription by the major nuclear PARPs, as well as the downstream physiological consequences of these regulatory events. Our broad hypothesis is that the gene regulatory activities of the nuclear PARPs are mediated through site-specific ADP-ribosylation of key regulatory proteins, such as histones and transcription factors. We will test this hypothesis using an integrated approach with a complementary of set tools from biochemistry, molecular biology, cell biology, chemical biology, proteomics, genomics, and mouse genetics. For these studies, we will focus on (1) Site- specific histone H2B ADP-ribosylation in pro-adipogenic signaling in preadipocytes, (2) C/EBP?-mediated pro- adipogenic signaling in preadipocytes, and (3) STAT1a-mediated interferon g (IFNg) signaling in macrophages. Our specific objectives are to: Characterize the genomic sites and substrates of nuclear PARP catalytic activity, with a focus on functional outcomes of histone ADP-ribosylation (Aim 1); Determine the effects of site- specific ADP-ribosylation on transcription factor function with a focus on C/EBP? in preadipocytes and STAT1? in macrophages (Aim 2); and Determine the effects of nuclear PARPs and ADP-ribosylation on physiological outcomes with a focus on adipocyte biology (Aim 3). Our proposed studies will shed new light on the molecular mechanisms underlying the control of signal- dependent gene expression by PARPs 1, 2, and 3 in preadipocytes and macrophages. They may also suggest better ways to target the nuclear PARPs to prevent, diagnose, and treat human metabolic and inflammatory diseases.